Probing Phospholipid Main Phase Transition by Fluorescence

Headgroup and acyl chain NBD-labeled phospholipid derivatives, viz. 1,2-dipalmitoyl-sn-glycero-3-phospho-[N-(4-nitrobenz-2-oxa-1,3-diazole)-ethanolami...
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11294

J. Phys. Chem. B 2001, 105, 11294-11301

Probing Phospholipid Main Phase Transition by Fluorescence Spectroscopy and a Surface Redox Reaction Juha-Matti I. Alakoskela and Paavo K. J. Kinnunen* Helsinki Biophysics and Biomembrane Group, Institute of Biomedicine/Biochemistry, UniVersity of Helsinki, Helsinki, Finland ReceiVed: March 22, 2001

Headgroup and acyl chain NBD-labeled phospholipid derivatives, viz. 1,2-dipalmitoyl-sn-glycero-3-phospho[N-(4-nitrobenz-2-oxa-1,3-diazole)-ethanolamine] (DPPN) and 1-acyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine (NBD-PC), were employed to study lipid conformations and organization in the course of the main phase transition of dipalmitoylglycerophosphocholine (DPPC) liposomes. The differential sensitivity of the 335 and 470 nm absorption bands to environment polarity was used to explore the ground-state NBD environment. Anisotropy measurements with excitation at those bands revealed the presence of two NBD-PC populations below Tm. The fluorescence maximum emission intensity, the ratio of emission intensities with excitation at 470 and 335 nm, and the rate coefficient k1 for the reduction of NBD by dithionite to nonfluorescent derivatives undergo complex changes in the course of the main phase transition of DPPC. For DPPN, the alterations in k1 and intensity ratio are likely to originate from a redistribution of this fluorophore at the transition and suggest a maximum in the bilayer transverse compressibility. For NBD-PC, the changes in these properties appear to be related to a shift in the fluorophore distribution at the percolation threshold for the two-phase region and self-quenching of the NBD moiety.

Introduction Phospholipids exhibit a great number of different lamellar and nonlamellar phases depending on temperature, pressure, and solvent environment.1,2,3 Perhaps the most thoroughly studied of the connecting transitions for phospholipids is the so-called main transition. The thermodynamic aspects of the phospholipid main phase transition are well understood,2 whereas less is known about the exact molecular mechanisms of the main phase transition. The main phase transition of lamellar lipid bilayers is considered to be a pseudocritical, weakly first-order process.4 The characteristic changes at the temperature (Tm) of the main phase transition from the solid, rippled gel-like Pβ′ phase to the fluidlike LR phase are the melting of the acyl chains (decrease in the chain conformational order) and the melting of the crystalline phospholipid lattice (decrease in the translational order). The entropy of the acyl chain melting2 drives the main phase transition, while changes in the polar region are considered secondary to those in the hydrocarbon region. Many properties of the phospholipid bilayers especially show discontinuities at Tm, such as membrane permeability12 and the lateral compressibility,13-16 which have maxima at Tm. These as well as other properties have been explained in terms of dominating critical density fluctuations leading to the formation of domains of geland fluid-state lipids, and further to maxima in the domain boundary length and the number of defects in membranes.4,12,17 However, recently it was suggested on the basis of fluorescence spectroscopic studies that this model might not be sufficient, for the domain boundary length appears to have a maximum * Address correspondence to: Dr. Paavo K. J. Kinnunen, Helsinki Biophysics and Biomembrane Group, Institute of Biomedicine/Biochemistry, Biomedicum, Haartmaninkatu 8, P.O. Box 63, FIN-00014 University of Helsinki, Finland. Fax: 358-9-19125444. E-mail: Paavo.Kinnunen@ Helsinki.fi.

already below the main phase transition temperature and may thus not account for the critical behavior.10 The bilayers of long acyl chains (from 17 to 20 methylene segments) phosphatidylcholines undergo in addition a so-called sub-main transition slightly below the respective temperatures of their main phase transition.5 The enthalpy for sub-main transition is very low and tends to zero with decreasing acyl chain lengths, whereas its transition temperature approaches the corresponding Tm with increasing acyl chain lengths. The sub-main transition has been suggested to be due to the uncoupling of the positional and configurational order, i.e., breakdown of the crystalline lattice order before the actual chain melting in the course of the main transition.6 Similar interpretations as well as mechanisms related to interfacial water organization have been suggested to explain the discontinuities observed slightly below Tm found in fluorescence, IR, and dielectric spectroscopy studies on dimyristoylphosphocholine (DMPC) and dipalmitoylphosphocholine (DPPC) liposomes.7-11 The knowledge of interrelations between different molecular events may prove useful when the findings suggesting the insufficiency of the present models7-11 and the possible need to redefine them are reviewed. The hydration and electric properties of the membrane-water interface in general have lately been gaining increasing attention and have been suggested to have a number of effects on biological systems.18-26 Changes in the movement and orientation of the headgroups are known to occur at the transition,27,28 and changes in the interfacial hydration water slightly below transition temperature have been reported.8,9 Both membrane dipole potential and zeta potential have been shown to decrease upon the main phase transition.29-33 On the basis of fluorescence spectroscopic studies with fluorescein-labeled lipid derivative, it has been suggested that the variations in the permittivity profile of the interface changes the sign of the dipole potential.34

10.1021/jp011080b CCC: $20.00 © 2001 American Chemical Society Published on Web 10/20/2001

Main Transition and NBD Fluorescence The change in the permittivity profile probably corresponds to alterations in the distribution and orientational polarizabilities of interfacial moieties, which in turn result from changes in the packing and freedom of movement of the lipid as well as water molecules. 7-Nitro-2,1,3-benzoxadiazol-4-yl (NBD) is a widely used fluorescent moiety in investigations on biological systems.36-41 Simple NBD derivatives have three major absorbance bands in the visible and near UV region, at approximately 420, 306360, and 225 nm.42,43 The lowest-energy absorption peak corresponds to the band of NBD-labeled lipids near 470 nm and is due to an intramolecular charge-transfer (ICT) type transition,43,44 which is associated with a large (∼4 D) change in the dipole moment.45 The 306-360 nm absorbance band (for NBD-lipids approximately 335 nm) corresponds to an ordinary π* r π transition.43 When exciting at either of these bands, the maximum emission wavelength λmax of NBD-labeled lipids lies at 520-535 nm, as expected on the basis of Kasha’s rule,46 while differences in fluorescence intensity correspond to the different absorptivities. The absorptivity of the 470 nm band and the quantum yield of fluorescence are both sensitive to the polarity and the hydrogen bonding capability of the environment and to the presence of charge transfer donors.42,43,47 When added to liposomes containing NBD-labeled lipids, either dithionite or its negatively charged radical form can rapidly reduce NBD to the nonfluorescent product, 7-amino2,1,3-benzoxadiazol-4-yl (ABD), thus allowing for the assessment of the content of the probe in the outer leaflet of the bilayer.48 Simultaneously with this rapid reaction, dithionite ion diffuses slowly across the bilayer, so as to reduce also the NBD groups in the inner leaflet. The reduction of NBD-labeled lipids by dithionite has been used, e.g., to study ion permeability of membranes undergoing phase transitions.12 The fluorescence characteristics of NBD-labeled lipids and the rate coefficients for the fast reduction of NBD located on the outer surface of liposomes were further shown to depend on the dipole potential of the membranes.35 In DPPN, the NBD moiety is linked to the headgroup, thus restricting its movements and maintaining it in the interface. In NBD-PC the chromophore is linked to the acyl chain; however, the chain may loop back so as to allow NBD to locate in the interface.49-51 We assessed both the spectral characteristics and dithionite reduction for these probes as a function of temperature in a DPPC matrix undergoing phase transition. We also introduce a novel way to exploit the steady-state fluorescence anisotropies and spectra of NBD to reveal the presence of different probe populations and to yield information on the environment of the ground-state NBD, respectively, without interference from excited-state photophysical or -chemical processes. Experimental Section Materials. DPPC was purchased from Sigma, Hepes was purchased from Boehringer Mannheim (Mannheim, Germany), and sodium dithionite, sodium carbonate, and sodium hydrogen carbonate were purchased from Merck (Darmstadt, Germany). NBD-PC was from Avanti Polar Lipids and DPPN from Molecular Probes (Eugene, OR). Stock solutions were prepared in chloroform, except for NBD-PC, which was dissolved in toluene:ethanol (1:1, by vol). The concentrations of the fluorescent lipids were determined spectrophotometrically using the molar absorptivities 463 ) 21 000 M-1 cm-1 (in CH3OH) and 465 ) 19 000 M-1 cm-1 (in C2H5OH) for DPPN and NBDPC, respectively. The concentration of DPPC was determined

J. Phys. Chem. B, Vol. 105, No. 45, 2001 11295 gravimetrically with a high precision electrobalance (Cahn Instruments, Inc., Cerritos, CA). Preparation of Liposomes. Appropriate amounts of the lipid stock solutions were mixed in chloroform, evaporated to dryness under a gentle nitrogen stream, and then maintained under reduced pressure for a minimum of one and a half hours to remove any residual solvent. The dry lipid residues were hydrated with 5.0 mM Hepes, pH 7.4, whereafter the suspensions were extensively vortexed, incubated for 30 min at 50 °C with continuous shaking, sonicated for 2 min with a bath sonicator, and incubated for another 30 min at 50 °C. To produce unilamellar vesicles, the hydrated lipid solutions were extruded with a LiposoFast pneumatic low-pressure homogenizer (Avestin, Ottawa, Canada) 19 times through Millipore (Bedford, MA) polycarbonate filters (pore size 100 nm) at a temperature well above the main phase transition temperature of the lipid. After extrusion, liposome solutions were kept on ice until used. Steady-State Fluorescence Measurements. The fluorescence emission spectra were recorded using a Perkin-Elmer LS50B spectrofluorometer with a magnetically stirred cuvette compartment thermostated with a circulating water bath (Haake C25, Haake, Germany). The temperature in the cuvette was measured with an immersed probe (HH42 Digital Thermometer, Omega Engineering, Inc., Stamford, CT). The excitation and emission band-passes were set at 5 nm. The emission anisotropy52 r was measured at 522 nm for DPPN and at 525 nm for NBD-PC, corresponding to their emission maxima, and the signal was averaged over a 10 s interval. Either 470 or 335 nm was used for excitation, as indicated, with each of the calculated spectra representing the average of five collected scans. The total lipid concentration in all steady-state fluorescence measurements was 100 µM, of which 3 mol % was either DPPN or NBD-PC. The measured maximum fluorescence intensities for DPPN and NBD-PC are expressed relative to the maximum fluorescence intensity of the respective probe at 45 °C. The measurement of the emission spectrum with excitation at 470 nm (I470) (used for the calculation of RFI) was immediately followed by the measurement of spectrum with excitation at 335 nm (I335), which was then used to obtain I470/I335. Accordingly, there should be only negligible error in the temperatures between corresponding RFI and I470/I335 measurements, and even minor differences in their response to temperature should be considered as significant. The absolute numeric values for I470/I335 appear to be somewhat sensitive to slight differences in the experimental conditions such as thermal history, or the presence of a small amount of multilamellar vesicles in the extruded samples. Stopped-Flow Fluorescence Spectroscopy. The kinetics of the reduction of NBD-labeled lipids by dithionite was measured using a stopped-flow spectrofluorometer (Olis RSM 1000F, OnLine Instruments, Inc., Bogart, GA) essentially as described previously.35 In brief, two pneumatically driven parallelly mounted syringes inject the reactants into the rapid mixing quartz glass fluorescence observation chamber. One of the syringes was loaded with a liposome solution (the total lipid concentration of 100 µM in 5.0 mM Hepes buffer, pH 7.4). The other syringe contained 10 mM dithionite in the buffer. The concentrations in the observation chamber were 5.0 mM for dithionite and 50 µM for lipid. The mole fraction X of the NBD-labeled lipids was 0.03 yielding 1.5 µM concentration of the indicated fluorescent marker. The excitation wavelength 470 nm was selected with a monochromator, and 21-62 spectra (473.5-626.5 nm) per second were measured. In keeping with previous findings,12,35 two independent first-order reactions provided the simplest model yielding good agreement with the

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measured data. Accordingly, software provided by the instrument manufacturer was used to fit the data by the equation

U ) A1e-k1t + A2e-k2t

(1)

where k1 and k2 represent the rate coefficients53 for the fast and slow components, respectively, A1 and A2 are the corresponding amplitudes, t represents time, and U is the photomultiplier tube output voltage. Results Effects on the Headgroup-Labeled DPPN. To investigate the changes in the bilayer surface upon the phospholipid main phase transition, we utilized the headgroup-labeled fluorescent phospholipid analogue DPPN. Three parameters were measured as a function of temperature for this probe in a DPPC matrix: relative emission intensity RFI (with λex ) 470 nm), the rate coefficient k1 for NBD reduction by dithionite, and I470/I335, i.e., the ratio of the emission intensities obtained at λex ) 470 and 335 nm, respectively. In the gel phase, RFI decreases slowly with increasing temperature until at approximately 37 °C emission intensity begins to increase, reaching a maximum at ∼41 °C, corresponding to Tm of DPPC (Figure 1A). Further increase in temperature results in a decrease in RFI. For the rate coefficient k1 for the reduction of NBD, the opposite behavior is observed. Accordingly, a slow increase in k1 is evident until at approximately 36 °C the values of k1 reach a maximum (Figure 1B). This is followed by a decrease in k1 to a minimum close to Tm, at ∼40 °C. Above Tm, an increase in temperature enhances the reaction rate. Arrhenius plots further demonstrate temperature dependence of k1 to be different for the gel and the fluid membranes, the apparent activation energy being almost 4-fold higher above Tm (Figure 1C). The plot of k1 versus RFI demonstrates that the temperature dependence of k1 and RFI are similar at temperatures distal from Tm (Figure 1F). Compared to the above parameters, I470/I335 shows a less complicated behavior (Figure 1D), decreasing with temperature and reaching a minimum at ∼40 °C. Further increase in T caused the I470/I335 ratio to increase. The fluorescence emission anisotropies with excitation at 335 and 470 nm (r335 and r470, respectively) as a function of T revealed the expected and previously reported decrease40 at the main transition temperature of DPPC (Figure 1E). Moreover, both r470 and r335 versus T have the same shape, the only difference being the slightly smaller values for the latter. Effects on the Chain-Labeled NBD-Lipid. The emission intensity as a function of temperature for NBD-PC in DPPC LUVs behaves very differently compared to DPPN. Starting at ∼20 °C, RFI first increases progressively with T, being approximately 3-fold at Tm, whereafter it progressively declines (Figure 2A). Distinctly different behavior from DPPN was also evident for the reduction of NBD-PC by dithionite, with an increase in temperature (from 20 to 50 °C) augmenting k1 from 0.13 to 1.05 s-1 (Figure 2B). The observed large scattering of the values for k1 measured for NBD-PC below Tm could originate from slightly different thermal histories. One possible reason for the slower reaction rates for DPPN (Figure 1B) compared to the neutral NBD-PC is the negative charge carried by the phosphate group of DPPN impeding the approach by the negatively charged dithionite. A further difference between the two probes is that unlike for DPPN (Figure 1C), the apparent activation energies for the reduction of NBD-PC by dithionite in both the gel and the fluid membranes are almost equal (Figure 2C). As expected on the basis of the S-shaped intensity curve

(Figure 2A) for NBD-PC, there is no correlation between k1 and RFI (Figure 2F). The intensity ratio I470/I335 increases at Tm, suggesting augmented apparent polarity of the environment of the chromophore of NBD-PC (Figure 2D). Fluorescence anisotropies r335 and r470 provide a more detailed view. Below Tm, the values for r335 are considerably higher than those for r470 (Figure 2E), while with T f Tm, the values for r335 approach those for r470. Above Tm, values for r335 are slightly lower than those for r470 (Figure 2E), similar to the data on DPPN over the whole temperature range (Figure 1E). Discussion In the temperature range of the main transition of DPPC liposomes, the k1 versus RFI dependence for the headgrouplabeled DPPN deviates from the linear relationship seen outside this region (Figure 1F). In brief, at the transition k1 is attenuated, while there is an increase in RFI, as well a minimum in I470/ I335. With increasing polarity of the environment the absorptivity of the ICT transition (at 470 nm) of NBD increases strongly, whereas the absorptivity of the π* r π transition (at 335 nm, to locally excited state) remains almost constant.43 The environment induced changes in the fluorescence quantum yields can be expected to be same for both excitation wavelengths. In a more polar environment, the increase in the absorptivity at 470 nm partly balances the enhanced nonradiative relaxation rate,43 and the lack of this effect for the absorptivity at 335 nm leads to a weaker fluorescence at λex ) 335 nm compared to excitation at λex ) 470 nm. With excitation at 335 nm, a larger proportion of the detected emission thus originates from the probes residing in a less polar environment. Accordingly, the ratio I470/I335 responds to the changes in the environment of the NBD in its ground state and should increase with increasing apparent polarity. The ground-state environment contains contributions mainly from the solvent relaxed state, whereas RFI includes the impact both from solvent dynamics and other excited-state photophysics. The decrease in the emission anisotropy of DPPN (Figure 1E) is in keeping with the reported discontinuities in the parameters describing the headgroup orientation and order in the main transition.27,55 As summarized above, by selecting the excitation wavelength of either 470 or 335 nm, the probe populations residing in different environmental polarities can be resolved. Accordingly, the presence of probe populations accommodated in sites of different polarities and apparent microviscosities can be detected without extensive spectral analysis by simply measuring the emission anisotropies r470 and r335 using excitation at 470 and 335 nm, respectively. The data processing is further simplified by the nearly equal fundamental anisotropy values at these excitation wavelengths. The almost equal r470 and r335 values for DPPN (Figure 1E) suggest that in the temperature range studied, there is absence of two probe populations for which both mobility and the polarity of surroundings are different, in contrast to those in NBD-PC (Figure 2E). The conformational distribution of the NBD moiety of the headgroup of DPPN is likely to be unimodal. In Langmuir-Blodgett films of gel-state monolayers of DPPC and DPPN, the average angle between the absorption dipole of NBD and the monolayer normal exceeds that measured for fluid domains,56 indicating the main transition to cause conformational changes in the DPPN headgroup. The temperature dependencies of the measured parameters for DPPN in DPPC are emphasized in Figure 3A, which depicts the temperature derivatives for RFI, k1, I470/I335, and r470. Since the derivative of r335 after scaling is almost same as that of r470 for both DPPN and NBD-PC, it has for the sake of clarity been

Main Transition and NBD Fluorescence

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Figure 1. Temperature dependence for RFI (A) and rate coefficient k1 for the reduction of DPPN (X ) 0.03) by dithionite (B) in DPPC liposomes. Fluorescence intensities are averages of two measuments. Rate coefficients represent averages from a total of six separate measuments (three measurements made for both of the two samplesof liposomes, except at temperatures 20.3 and 23.6 °C, at which three measurements of one sample were made). The error bars represent standard deviation. Panel C depicts Arrhenius plots for the temperature ranges below and above (marked by 1 and 2, respectively) the discontinuity at the transition. Arrhenius parameters below Tm are A ) 2.3 × 106 s-1 and Ea ) 22 kJ/mol and, above Tm, A ) 1.2 × 1028 s-1 and Ea ) 80 kJ/mol. Panel D shows the changes in I470/I335. Data were normalized by setting the maximum and minimum values at one and zero, respectively. The data points are averages of two experiments, in which the actual intensity ratio ranged between 2.94-3.06 and 2.98-3.12. Panel E shows the values for the anisotropy r measured using excitation at 470 nm (9, r470) and 335 nm (O, r335). Panel F illustrates compilation of the data as k1 vs RFI. Complete description of the experiments is given in Materials and Methods.

omitted from this figure. The temperature differences of the changes in k1, RFI, and I470/I335 in the transition region suggest that they correspond to different processes involved either in the transition itself6,10,11 or processes such as decreased selfquenching of the probe and alterations in the apparent environmental polarity. The steepest slope in RFI versus T, i.e. the maximum in the d(RFI)/dT, is observed at approximately 39.9 °C. At approximately the same temperature, both d[I470/I335]/ dT ≈ 0 and d[k1]/dT ≈ 0 (39.9 and 40.1 °C, respectively); i.e., the minima in I470/I335 versus T and k1 versus T coincide with the steepest slope in RFI versys T. The maximum in RFI is observed at 40.8 °C. Although in DPPC monolayers DPPN

partitions almost equally into the coexisting gel and fluid phases37 due to electrostatic repulsion, the increase in RFI (Figure 1A) could be related to an increased number of DPPN molecules becoming dispersed into the fluid phase by breakdown of DPPN-enriched domains, thus leading to a concomitant decrease in self-quenching. Aggregation and self-quenching of the probe in the gel phase is supported by analysis of the fluorescence lifetimes for a headgroup-labeled NBD lipid,58 and by the decrease in the relative magnitude of the peak in RFI at Tm when the mole fraction of the probe in DMPC liposomes is decreased.38 The maximum in d(RFI)/dT would be in keeping with most efficient breakdown of the DPPN-enriched domains

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Figure 2. Behavior of the chain-labeled fluorophore NBD-PC (X ) 0.03) in DPPC matrix. Rate coefficients represent averages of a total of 9-14 measurements for four separate samples. Fluorescence intensities are averages of four and anisotropies of two measurements. In all panels error, bars indicate standard deviations. (A) The relative maximum fluorescence intensity RFI and (B) the rate coefficient k1 as a function of temperature. (C) Arrhenius plots for temperatures below and above (marked by 1 and 2, respectively) the main phase transition. Arrhenius parameters below Tm are A ) 3.4 × 109 s-1 and Ea ) 59 kJ/mol and, above Tm, A ) 3.1 × 1010 s-1 and Ea ) 65 kJ/mol. (D) The ratio of emission intensities with excitation at 470 or 335 nm. Data were normalized by setting the maximum and minimum values at one and zero, respectively. The values shown represent the averages from four experiments, in which the actual intensity ratio ranged between 2.77-2.92, 2.79-2.91, 2.05-2.26, and 2.993.21. (E) The fluorescence anisotropies vs temperature acquired using excitation at 470 nm (9) and 335 nm (O). (F) Compilation of the data as k1 vs RFI.

at the same temperature at which k1 and I470/I335 have their minima. This temperature is likely to represent Tm. The decrease in k1 near Tm (Figure 1B) complies with dynamic membrane lateral heterogeneity.17 If the negatively charged DPPN molecules were transiently enriched into microdomains while the membrane is undergoing transition,10 the surface charge density of those transient domains would be higher,11 and the rate of reduction by the negatively charged dithionite could be expected to decrease. Yet this possibility would also require enhanced self-quenching of the DPPN and is thus in contradiction with the peak in RFI (Figure 1A). Accordingly, a transient formation

of DPPN-enriched microdomains is unlikely to explain the decrease in k1 at the transition. From a ζ potential study assessing temperature and ionic strength dependence of cation binding, it was concluded that the positively charged choline headgroup resides deepest inside the membrane and the phosphate group at the outer layer more exposed to the water phase at Tm.28 In keeping with the above, the decrease in k1 and the minimum in I470/I335 at the transition both suggest that also the NBD moiety becomes immersed deeper in the membrane in the transition region. If the movement of the headgroups was the cause for changes seen in I470/I335

Main Transition and NBD Fluorescence

Figure 3. The temperature derivatives (D) of RFI, k1, I470/I335, and r470 vs T. To accommodate these data into a single panel, all the derivatives were scaled by dividing each value by the difference between largest and smallest value of that derivative. The horizontal line is set at D ) 0. (A) The numerical derivatives for the above properties for DPPN in DPPC vesicles, RFI (9, solid line), k1 (1, dotted line), I470/I335 (O, solid line), and r470 (], dotted line). (B) The numerical derivatives for NBD-PC in DPPC vesicles, RFI (9, solid line), k1 (1, dotted line), I470/I335 (O, solid line), and r470 (], dotted line). While all the other numerical differentiates presented in panels were calculated without prior smoothing of the data, due to high noise level, the calculation of d(k1)/dT vs T was preceded by adjacent averaging of three points of the k1 vs T data. Accordingly, the apparent decrease for the last data point (at 49.6 °C) is an artifact produced by smoothing.

and k1, opposite changes above Tm are to be expected on the basis of the headgroup conformations suggested by Makino et al.28 However, a model reproducing the characteristics of measured NMR parameters estimates the average angle between the P-N dipole of phosphocholine and membrane plane to be 30° in the gel phase, and 0-3° in the fluid phase.59 Accordingly, the orientation of choline back toward the water phase is probably not complete. The fluid phase also exhibits headgroup fluctuations by 4-5 Å perpendicular to the bilayer plane.59 The different orientations and headgroup fluctuations could contribute to the different temperature dependence (apparent activation energy) of the rate coefficient of the reduction in the fluid and gel phases (Figure 1C). As discussed above, the changes in both k1 and I470/I335 for DPPN in the transition region are in keeping with the NBD moiety being transiently located in a more hydrophobic environment. More specifically, in the transition region the access of dithionite to the NBD moiety of DPPN is impeded. In the twophase region, the lateral compressibility of the bilayer has a maximum.13-16 Our data suggest that in the transition region,

J. Phys. Chem. B, Vol. 105, No. 45, 2001 11299 there is a maximum also in membrane transverse compressibility. Accordingly, this would allow the dipole potential of the membrane to pull the DPPN headgroup with its negative charge slightly deeper into the membrane. The coupling between the lateral and the transverse or thickness compressibilities is implicitly used in some studies on the squeeze mode thickness fluctuations, justified with the liquid nature of lipid bilayers and consequent to small volume compressibility.60 The relatively small ∼5% change in bilayer volume compared to the 20% area change and the 15% decrement in thickness at the main transition also necessitates a loose coupling of the lateral and transverse or thickness compressibilities at the transition.2 Combined maxima in both the transverse and the lateral compressibility could thus allow the NBD moiety to become accommodated deeper into the bilayer with respect to the phosphocholine headgroups. This further implies the extent of the trans f gauche isomerization of the DPPN chains to slightly exceed that for the surrounding DPPC. A behavior very different from that of DPPN is observed in the transition region for the acyl chain-labeled NBD-PC. To allow for a comparison with the data on DPPN, the numerical temperature derivatives are presented in Figure 3B. Except for the d[I470/I335]/dT versus T, they are similar to the respective derivatives for DPPN. For NBD-PC, the most pronounced changes in RFI, I470/I335, and r470 coincide at 40.3 °C. Because of only minor changes being evident in k1 at Tm, its derivative warrants no further discussion. The large increase in RFI observed for NBD-PC at the transition (Figure 2A) is readily explained by the breaking of microdomains enriched in the fluorophore and the concomitant decrease in self-quenching.54 In keeping with this, the propensity of NBD-PC to segregate into microdomains in the gel state membranes and to partition into the fluid domains in the fluid/gel coexistence region is wellknown from epifluorescence studies on monolayers.37 For NBDPC, there seems to be only a minor plateau in k1 versus T in the transition region (Figure 2B) instead of the major discontinuity seen for DPPN (Figure 1B). Also, the apparent activation energies for k1 below and above Tm are almost equal (Figure 2C). This suggests that with regard to the reduction of the NBD by dithionite, the fluorophore moiety of NBD-PC appears similar both below and above Tm and supports an enrichment of NBDPC into fluidlike microdomains below Tm. This is further supported by our anisotropy measurements (Figure 2E). For the fraction of NBD-PC in a more polar environment (as estimated from the r470), the transition seems to be diffuse, especially when taking into account that the steeper anisotropy change for the probe in a more rigid and less polar environment (seen in r335) also contributes to r470. For DPPN (Figure 1E) and for the more rigid population of NBD-PC with the fluorophore in a less polar environment (r335), there is sharp increase in the probe mobility at the transition (Figure 2E). Again, the more diffuse transition must smear out the signal for sharper transition, as the excitation wavelength allows only a weighing between the signals from different populations, not absolute selection. The larger values of r335 compared to those of r470 below Tm suggest that a fraction of NBD moieties resides in a less polar and more rigid environment, which is likely to be the acyl chain region. Studies suggesting the chromophore of NBD-PC to partition only within the interfacial region36,49 were conducted using a fluid phase matrix. The intensity ratio I470/I335 displays a steep increase at the transition suggesting that for a fraction of the NBD dye the polarity of the environment increases sharply (Figure 2D). A likely explanation is that due to chain reversal,49,61 increasing parts of the NBD moieties initially located within the acyl chain

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region become accommodated into the interfacial region. This is also supported by our anisotropy measurements, as the values for r335 decrease below the values of r470 close to the Tm (Figure 2E), thus evidencing the disappearance of the NBD population in a less polar and more rigid environment. This could relate to the percolation threshold. More specifically, NBD-PC in a conformation with chain reversal and with the NBD moiety thus accommodated within the interface requires a larger projected cross-sectional area in the plane of the membrane. Below the percolation threshold, chain reversal for NBD-PC in fluid domains would exert pressure on the surrounding continuous gel phase lattice. At the percolation threshold, the fluid phase becomes continuous, which increases lateral compressibility and reduces the energy barrier for chain reversal, thus allowing for a shift in the conformational equilibrium of NBD-PC toward chain reversal, bringing the NBD moiety into the interfacial region. Except for the changes in r470 and r335 related to domains and the vertical distribution of the fluorophore moiety of NBDPC, the values for r335 are consistently slightly smaller than those for r470 for both DPPN and NBD-PC (Figure 1E, Figure 2E). An adequate explanation could be provided either by a small angle between the absorption dipole moments for the 335 and 470 nm transitions leading to different fundamental anisotropies or by a difference in the lifetime due to relaxation processes from the 335 nm excited state into the 470 nm excited state. Assuming that only one of the above two factors contributes at a time allows one to estimate the difference needed for the observed effects in these parameters. This is based on the following equations:

r0 )

(

)

2 3 cos2 β - 1 5 2

(2)

where r0 is fundamental anisotropy, τ is fluorescence lifetime,

r0 τ )1+ r θ

(3)

and θ is rotational correlation time for 3D movements of a sphere.52 The value for the angle β between the absorption and emission dipoles of NBD has been measured to be approximately 25° for the excitation at 470 nm.56 Accordingly, a difference of 1.00° ( 0.65° in β or 8.5 ( 6.0% in lifetime would be sufficient to cause the differences in anisotropy. Very small difference in β seems more plausible. Conclusions Comparison of the fluorescence properties and reduction of the NBD moiety in the headgroup and chain-labeled lipid analogues DPPN and NBD-PC, respectively, to probe the main transition of DPPC revealed a number of novel features. Studies with DPPN suggest that concomitantly with the maximum in lateral compressibility of the bilayer also the transverse compressibility is augmented close to Tm. Accordingly, in the main transition region, the NBD moiety of DPPN becomes transiently immersed deeper into the more hydrophobic part of the bilayer, impeding its reduction by dithionite and decreasing the ratio of fluorescence intensities acquired with different excitation wavelengths. The changes in the fluorescence intensity ratio and anisotropies measured with different excitation wavelengths suggest a change in the vertical distribution of the chromophore moieties of NBD-PC at the percolation threshold in a manner indicating a first-order process. More specifically, as the fluid phase becomes continuous at the percolation threshold, the

restraint exerted by the gel phase lattice to the entrapped fluid domains diminishes and allows augmented acyl chain reversal, which brings the attached NBD moiety into the interface, together with a simultaneous increase in the fluid phase interfacial area. Abbreviations A ABD DMPC DPPC DPPN Ea Hepes ICT k1 NBD NBD-PC r335 r470 RFI

β λex λmax

apparent frequency factor 7-amino-2,1,3-benzoxadiazol-4-yl 1,2-dimyristoyl-sn-glycero-3-phosphocholine 1,2-dipalmitoyl-sn-glycero-3-phosphocholine 1,2-dipalmitoyl-sn-glycero-3-phospho-[N-(4-nitrobenz-2oxa-1,3-diazole)-ethanolamine] apparent activation energy (N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]) internal charge transfer rate coefficient for fast reaction 7-nitro-2,1,3-benzoxadiazol-4-yl 1-acyl-2-[12-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]dodecanoyl]-sn-glycero-3-phosphocholine fluorescence anisotropy acquired using excitation wavelength 335 nm fluorescence anisotropy acquired using excitation wavelength 470 nm maximum fluorescence intensity relative to maximum fluorescence intensity of DPPN or NBD-PC in DPPC liposomes at 45 °C the angle between the absorption and emission transition dipoles of NBD excitation wavelength maximum fluorescence emission wavelength

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